Airway disease is the major cause of morbidity and mortality in cystic fibrosis (CF), an autosomal recessive disease caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel. Welsh et al., The Metabolic and Molecular Basis of Inherited Disease, eds. Scriver CR, Beaudet AL, Sly WS, Valle D, Childs B, Vogelstein B, McGraw-Hill, New York, 1995. Gene transfer offers the potential for a new and effective treatment for CF airway disease. For reviews see Davies et al., 2001, J. Gene Med. 3:409-417; Flotte, 1999, Curr. Opin Mol. Ther. 1:510-516; and Welsh, 1999, J. Clin. Invest. 104:1165-1166. Previous studies have shown the feasibility of transferring the CFTR cDNA to CF airway epithelial cells in vitro and in vivo. However, with most vectors, two main problems limit gene transfer: gene transfer across the apical surface of differentiated airway epithelia is inefficient, and expression of the transferred gene is transient. See Davies et al., 2001, J. Gene Med. 3:409-417; Flotte, 1999, Curr. Opin Mol. Ther. 1:510-516; and Welsh, 1999, J. Clin. Invest. 104:1165-1166.
Adeno-associated virus (AAV) vectors offer several potential advantages as vectors for the transfer of the CFTR gene to CF airway epithelial cells. First, they have an excellent safety record in the lab and in humans. Second, they target both dividing and non-dividing cells like those in airway epithelia. Third, they do not induce a cell-mediated immune response. Fourth, they have been reported to generate long-term transgene expression. Fifth, unlike adenovirus and other human AAV serotypes, serotype 5 of human AAV (AAV5) targets the apical surface of differentiated airway epithelia. See Zabner et al., 2000, J. Virol. 74:3852-3858.
The utility of AAV vectors for CF gene transfer, however, is limited by their packaging capacity. The single-stranded genome of AAV5 is 4642 bp in length, which is similar to that of other AAV serotypes (Chiorini et al., 1999, J. Virol. 73:1309-1319), making it likely that AAV vectors will package only relatively small genomes. The only cis components required for replication and packaging of the recombinant genome into AAV5 virions are the two AAV5 ITRs, each 167 bp in length. Rabinowitz et al., 2000, Virology 278:301-308. The full-length CFTR cDNA is 4443 bp in length from the ATG through the stop codon. Thus, the length of the ITRs and a full-length CFTR cDNA (4777 bp) exceeds the length of the wild-type AAV5 genome. Moreover, an AAV expression cassette must also include a promoter, an intervening sequence (IVS) between the transcription and translation start sites, and a poly(A) addition sequence. See FIG. 1 for a schematic representation showing a typical arrangement of these elements in an AAV expression cassette.
Dong et al. studied DNA of various sizes and concluded that the optimal packaging limit for the AAV2 serotype was 4.9 kb; above this limit, packaging efficiency dropped precipitously. Dong et al., 1996, Human Gene Therapy 7:2102-2112. This observation is consistent with the previous findings that AAV vectors containing transgene inserts substantially longer than 4.9 kb have been difficult to produce.
In general, vectors that contain cDNA encoding truncated CFTR with short promoters or encoding full-length CFTR with no promoter other than the ITR were packaged with varying efficiency. See Flotte et al., 1993, J. Biol. Chem. 268:3791-3790; Zhang et al., 1998, Proc. Natl. Acad. Sci. USA 95:10158-10163; Wang et al., 1999, Gene Therapy 6:667-675. Recombinant vectors longer than 4900 bp were packaged less efficiently or not at all.
For example, Flotte et al. found that a 5010 bp DNA encoding CFTR was not packaged. However, they were able to package a 4647 bp vector that contained cDNA encoding CFTR with residues 1-118 deleted. In this vector, the ITRs were used as a promoter. Flotte et al., 1993, J. Biol. Chem. 268:3791-3790. Similarly, Zhang et al. reported that they could package a 4837 bp sequence containing a full-length CFTR cDNA with no promoter other than the ITRs, but the vector generated no CFTR Cl-current. They also reported that a 4727 bp cassette with a p5 promoter and a CFTR containing deletions in both the C-terminus and the R domain generated Cl− current, as detected by the very sensitive patch-clamp technique in isolated cells. Zhang et al., 1998, Proc. Natl. Acad. Sci. USA 95:10158-10163. Wang et al. also produced a 4983 bp AAV vector with CFTR under control of the p5 promoter and reported detectable CFTR expression by whole-cell patch clamp in JME CF cells, but that genome was packaged much less efficiently than the 4837 bp genome of Zhang et al. Wang et al., 1999, Gene Therapy 6:667-675.
From these studies, it is clear that the relatively small packaging size limit of AAV vectors places severe constraints on the generation of AAV-based vectors for transfer of the CFTR cDNA. There are no reports of AAV-based vectors containing CFTR-encoding constructs longer than 5 kb. There are some reports of limited packaging into AAV virions for CFTR constructs of 4.9 and 5 kb in length. However, evidence that CFTR protein was expressed in cells transduced by these vectors relied on very sensitive patch-clamp detection techniques in single cells, and there was no evidence that expression was sufficient to generate trans-epithelial Cl− current in an epithelium.
The longest components contained within the AAV expression cassette (FIG. 1) are usually the promoter and the transgene. Thus, the two most likely ways in which the length of the expression cassette may be reduced would be to shorten the promoter or to shorten the transgene. The coding sequence of full length CFTR is 4450 bp. Riordan et al., 1989, Science 245:1066-1073. Addition of the two inverted terminal repeats of AAV (300 bp), and minimal 3′ and 5′ untranslated regions (˜100 bp) yields an insert (4850 bp), which leaves little room for enhancer-promoter elements, most of which are >600 bp. However, several groups have shown that selective deletion of portions of the coding region of a gene can decrease the overall size of the gene while still allowing expression of an active protein molecule. This approach has been successfully employed to create a mini-dystrophin gene for use in gene therapy for Duchenne muscular dystrophy (DMD; Phelps et al., 1995, Hum. Mol. Genet. 4:1251-1258) and also for CFTR (Zhang et al., 1998, Proc. Natl. Acad. Sci. 95:10158-10163; and Flotte et al., 1993, J. Biol. Chem. 268:3781-3790).
The R (regulatory) domain of the CFTR protein extends approximately from residues 634-708 at the N-terminus to approximately 835 at the C-terminus. See Ostedgaard et al., 2001, J. Biol. Chem. 276:7689-7692; Ostedgaard, et al., 2000, Proc. Natl. Acad. Sci. U.S.A. 97:5657-5662; and Csandy et al., 2000, J. Gen. Physiol. 116:477-500. Previous work has shown that a peptide encompassing residues 708-831 regulates activity, but in solution forms a predominantly random coil. Ostedgaard, et al., 2000, Proc. Natl. Acad. Sci. U.S.A. 97:5657-5662.
Several earlier studies showed that CFTR molecules in which portions of the R domain had been deleted still retained some CFTR function as a chloride ion channel. See Rich et al., 1991, Science 253:205-207; Rich et al., 1993, Receptors Channels 1:221-232; Ma et al., 1997, J. Biol. Chem. 272:28133-28141; Zhang et al., 1998, Proc. Natl. Acad. Sci. U.S.A. 95:10158-10163; Vankeerberghen et al., 1999, Biochemistry 38:14988-14998; and Xie et al., 2000, Biophys. J. 78:1293-1305. However, at least some of these deletions induced channel activity in the absence of phosphorylation, reduced the response to PKA-dependent phosphorylation, and/or reduced net channel activity. See Rich et al., 1991, Science 253:205-207; Rich et al., 1993, Receptors Channels 1:221-232; Ma et al., 1997, J. Biol. Chem. 272:28133-28141; Zhang et al., 1998, Proc. Natl. Acad. Sci. U.S.A. 95:10158-10163; Vankeerberghen et al., 1999, Biochemistry 38:14988-14998; Xie et al., 2000, Biophys. J. 78:1293-1305; and Ostedgaard et al. 2001, J. Biol. Chem. 276:7689-7692. Moreover, previous studies have only examined CFTR expressed in heterologous cell lines and studied activity using the patch-clamp technique, planar lipid bilayers, or anion efflux. There was little information about their function in airway or other epithelia, which is critical in assessing the value of these proteins in gene transfer applications because deletions could alter protein-protein interactions, targeting to the apical membrane, constitutive and stimulated activity, phosphorylation-dependent regulation, and perhaps toxicity.
In contrast, Ostedgaard et al. have developed a shortened CFTR transgene (CFTR-ΔR) in which biosynthesis, localization, and Cl− channel function of this CFTR-ΔR protein were demonstrated to be the same as wild-type CFTR in airway epithelia. Ostedgaard et al., 2002, Proc. Natl. Acad. Sci. USA 99:3093-3098. In these studies, however, an adenoviral vector, which has a much greater packaging capacity than AAV-based vectors, was employed to transfer the DNA sequence encoding the CFTR-ΔR into the airway epithelial cells. Incorporation of the same CFTR-ΔR expression cassette employed by Ostedgaard et al. into an AAV vector would still impose some packaging limitations. Thus, some truncation of the promoter sequence would still be necessary to achieve efficient rescue of this expression cassette in AAV vectors.
The cytomegalovirus immediate early (CMVie) enhancer-promoter is one of the most widely-employed promoters in gene transfer vectors. See Stinski, 1999, In Gene Expression Systems: Using Nature for the Art of Expression. Academic Press, New York. 1999. pp. 211-233. The CMVie enhancer-promoter directs expression in many different cell types, generates higher levels of expression than most other enhancer-promoters, and functions in many viral and non-viral vectors. For example, in the airway epithelial lines ELM and CFT1, the CMVie enhancer-promoter generated much greater expression of a reporter gene than promoters of a housekeeping gene (ubiquitin B), a cytokine gene (interleukin 8), a signaling ligand gene (nitric oxide synthase; NOS), the tissue-specific genes MUC1, CC10 and SPC, or another viral promoter (adenovirus E1a). Yew et al., 1997, Human Gene Therapy 8:575-584. Importantly, this relative expression pattern also was observed in vivo in mouse lung, and CMVie enhancer-promoter drives CFTR expression and corrects the CF Cl− transport defect in cultured airway cell lines (JME/CF15) and primary cultures of differentiated airway epithelia. See Ostedgaard et al, 2002, Proc. Natl. Acad. Sci. USA 99:3093-3098; Jiang et al., 1996, Am. J. Physiol. 271:L527-L537. Forms of the CMVie enhancer truncated at nt −348 or nt −222 were observed to retain some activity. Stinski and Roehr, 1985, J. Virol. 55:431-441.